Journal: Biology Open

Article Title: The anti-motility signaling mechanism of TGFβ3 that controls cell traffic during skin wound healing

doi: 10.1242/bio.20122246

Figure Lengend Snippet: Serum-starved HDFs and HKCs in duplicate culture plates were either unstimulated or stimulated with TGFβ3 (3 ng/ml) for 10 min. The stimulation was stopped by removing the medium from the plates and rinsing of the plates three times with ice-cold PBS solution on ice. The cells were then subjected to Protein Kinase Array screening strictly following the manufacturer's procedures (Materials and Methods), including lysing cells with a special lysis buffer provided. ( A ) Images of ECL-developed films, where antibody identities on the spots were based on the information given by the manufacturer. ( B , C ) Phosphoimager quantitation of phospho-ERK, phospho-CREB and phospho-Src in HDFs and HKCs. ( D ) Stronger activation of PKA pathway, i.e. phosophorylation of CREB, in HDFs than in HKCs by TGFβ3 (10 min) was confirmed by Western blots (lane 2 versus lane 4). ( E ) Downregulation of Smad4 blocks TGFβ3-induced CREB phosphorylation (lane 4 versus lane 2). The Array experiment was repeated twice and the Western blotting experiment four times. Similar results were obtained.

Article Snippet: The third or fourth passages of cells were used for experiments. rhPDGF-bb, rhTGFα, rhTGFβ3, anti-phospho-CREB (Ser-133) antibody and anti-Smad4 antibody were purchased from R&D System (Minneapolis, MN).

Techniques: Lysis, Quantitation Assay, Activation Assay, Western Blot, Phospho-proteomics

Journal: Biology Open

Article Title: The anti-motility signaling mechanism of TGFβ3 that controls cell traffic during skin wound healing

doi: 10.1242/bio.20122246

Figure Lengend Snippet: ( A ) HDFs were serum starved overnight and subjected to colloidal gold migration assays in the absence or presence of PDGF-bb (15 ng/ml), TGFβ3 (3 ng/ml) and PKA inhibitor, H89 (1.0 µM). Representative images of migrated cells under the indicated conditions are shown (a to d). Quantitative analyses of the migration tracks are shown underneath as Migration index (MI) (%). Average size migration tracks are marked with dotted circles. This experiment was repeated multiple times and similar results were obtained. ( B ) Serum-starved HDFs were treated without or with TGFβ3 or TGFβ3 plus H89. Total cell lysates were subjected to immunoblotting analyses with indicated antibodies. ( C ) Serum-starved HDFs were subjected to colloidal gold migration assay, as previously described, in the absence or presence of PDGF-bb (bar 1 and 2) or PDGF-bb plus increasing concentrations of forskolin, an activator of PKA (bars 3 to 7). The effectiveness of forskolin (10.0 µM) on HDFs is indicated by causing an increased phosphorylation of CREB (insert image) in the cells. These experiments were repeated three times. *Statistically significant over the control, P <0.05.

Article Snippet: The third or fourth passages of cells were used for experiments. rhPDGF-bb, rhTGFα, rhTGFβ3, anti-phospho-CREB (Ser-133) antibody and anti-Smad4 antibody were purchased from R&D System (Minneapolis, MN).

Techniques: Migration, Western Blot, Phospho-proteomics, Control

Journal: Biology Open

Article Title: The anti-motility signaling mechanism of TGFβ3 that controls cell traffic during skin wound healing

doi: 10.1242/bio.20122246

Figure Lengend Snippet: ( A ) HDFs were infected with FG-12 lentivirus carrying either a GFP control gene or a shRNAs against PKA-Cα. After 48 hours, downregulation of the endogenous PKA-Cα was confirmed by immunoblotting the lysates of the cells with an anti-PKA-Cα antibody. ( B ) Downregulation of PKA-Cα blocks forskolin-stimulated CREB phosphorylation (lane 4 versus lane 2). ( C ) Downregulation of PKA-Cα showed little effect on TGFβ3-stimulated phosphorylation of Smad3 (lanes 4 and 6 versus lane 2). ( D ) Two sets of PKA-Cα-downregulated HDFs were subjected to colloidal gold migration assay in response to PDGF-bb (15 ng/ml) in the absence or presence of TGFβ3 (3 ng/ml). Representative images of the migrated cells under the indicated conditions are shown (a to i). ( E ) The computer-assisted quantitative analyses of the migration tracks are shown as Migration index (MI) (%). The experiment was repeated four times ( n = 3, P ≤0.05). *Statistically significant over the control.

Article Snippet: The third or fourth passages of cells were used for experiments. rhPDGF-bb, rhTGFα, rhTGFβ3, anti-phospho-CREB (Ser-133) antibody and anti-Smad4 antibody were purchased from R&D System (Minneapolis, MN).

Techniques: Infection, Control, Western Blot, Phospho-proteomics, Migration

Journal: The Tohoku journal of experimental medicine

Article Title: Low-intensity aerobic exercise mitigates exercise-induced bronchoconstriction by improving the function of adrenal medullary chromaffin cells in asthmatic rats.

doi: 10.1620/tjem.234.99

Figure Lengend Snippet: Fig. 5. Low- or moderate-intensity aerobic exercise inhibits the ERK/CREB signaling and downstream gene expression in rat adrenal medulla. The relative levels of phosphorylated ERK and CREB were characterized by Western blot, and the relative levels of c-FOS, EGFR1, c-JUN, JUNB, and FOSB mRNAs to the control β-actin in the adrenal medullary tissues of individual rats were characterized by qRT-PCR. Data are representative images or expressed as the mean ± s.d. of individual groups of rats (n = 4 per group) from three separate experiments. (A) The relative levels of phosphorylated ERK and CREB. (B) The relative levels of c-FOS, EGFR1, c-JUN, JUNB, and FOSB mRNAs. Control: Control rats; Low: The rats with low-intensity aerobic exercise; Mod: The rats with moderate-intensity aerobic exercise; OVA: The rats were sensitized and challenged with OVA; OVA + Low: The rats were sensitized and challenged with OVA and subjected to low-intensity aerobic exercise; OVA + Mod: The rats were sensitized and challenged with OVA and subjected to moder- ate-intensity aerobic exercise. *P < 0.05 vs. the control group; ▲P < 0.05 vs. the OVA group; #P < 0.05 vs. the OVA + Low group.

Article Snippet: The animals were sacrificed and studied on day 56. incubated with anti-peripherin (1:20,000), anti-neurofilament-68 (NF68, 1:5,000), anti-chromogranin A (CgA, 1:10,000), anti-PNMT (1:1,000), anti-ERK1/2 (extracellular signal-regulated kinase, 1:1,000), anti-phosphorylated ERK1/2 (anti-p-ERK1/2, 1:500, Abcam, Cambridge, UK), anti-CREB (cAMP responsive element binding protein, 1:1,000), anti-phosphorylated CREB (anti-p-CREB, 1:1,000), or anti-β-actin (1:1,000, Cell Signaling Technology, Beverly, USA) overnight at 4°C.

Techniques: Gene Expression, Western Blot, Control, Quantitative RT-PCR

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Novel mechanisms and signaling pathways of esophageal ulcer healing: the role of prostaglandin EP2 receptors, cAMP, and pCREB

doi: 10.1152/ajpgi.00177.2014

Figure Lengend Snippet: cAMP response element-binding protein (CREB) expression and phosphorylation (pCREB) in normal and ulcerated esophageal tissues. A, top: Western blot analyses of pCREB and total CREB (phosphorylated + unphosphorylated) protein expression in normal and ulcerated esophageal tissue 3, 6, and 9 days after ulcer induction. Esophageal ulceration induced phosphorylation of CREB. Bottom: quantitative analysis of pCREB. B, top: Western blot analyses of pCREB and total CREB protein expression in esophagus of rats treated intragastrically with either a single 50 μg/kg dose of misoprostol (MS) or its vehicle (VH). Misoprostol treatment further increased pCREB induced by esophageal ulceration. Bottom: quantitative analysis of pCREB. CREB phosphorylation was determined by calculating the ratio between pCREB and total CREB protein levels. Values are means ± SD. For each column (n = 6).

Article Snippet: The membranes were incubated with rabbit polyclonal anti-EP1, anti-EP2, anti-EP3, or anti-EP4 antibodies (Cayman Chemical, Ann Arbor, MI), mouse monoclonal anti-phosphorylated CREB (pCREB) (Cell Signaling Technology, Beverly, MA), or anti-VEGF (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies at room temperature for 1 h. Membranes probed with pCREB were stripped and reprobed using rabbit polyclonal anti-CREB antibody (Santa Cruz Biotechnology), which recognizes both pCREB and unphosphorylated CREB.

Techniques: Binding Assay, Expressing, Phospho-proteomics, Western Blot

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Novel mechanisms and signaling pathways of esophageal ulcer healing: the role of prostaglandin EP2 receptors, cAMP, and pCREB

doi: 10.1152/ajpgi.00177.2014

Figure Lengend Snippet: cAMP mediates misoprostol-induced CREB phosphorylation and stimulation of VEGF expression in HET-1A cells. Cells were treated with either the cAMP analog Sp-cAMP (100 μmol), misoprostol (10 μmol), or PBS for 30 min (A) or 3 h (B). Misoprostol-treated cells were pretreated with either PBS or inhibitor of cAMP-dependent PKA, Rp-cAMP (500 μmol) for 30 min. A, top: Western blot analyses of pCREB and total CREB (phosphorylated + unphosphorylated) protein expression. Sp-cAMP induced CREB phosphorylation in HET-1A cells, and Rp-cAMP inhibited this effect. Bottom: quantitative analysis of CREB phosphorylation. CREB phosphorylation was determined by calculating the ratio between pCREB and total CREB protein levels. B, top: RT-PCR analysis of VEGF mRNA expression. Sp-cAMP induced VEGF mRNA expression in HET-1A cells, and Rp-cAMP inhibited this effect. Bottom: quantitative analysis of VEGF mRNA expression. Each signal was normalized against the corresponding β-actin signal, and the results are expressed as VEGF/β-actin. All values are means ± SD of 3 separate experiments performed in duplicate.

Article Snippet: The membranes were incubated with rabbit polyclonal anti-EP1, anti-EP2, anti-EP3, or anti-EP4 antibodies (Cayman Chemical, Ann Arbor, MI), mouse monoclonal anti-phosphorylated CREB (pCREB) (Cell Signaling Technology, Beverly, MA), or anti-VEGF (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies at room temperature for 1 h. Membranes probed with pCREB were stripped and reprobed using rabbit polyclonal anti-CREB antibody (Santa Cruz Biotechnology), which recognizes both pCREB and unphosphorylated CREB.

Techniques: Phospho-proteomics, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Bone

Article Title: Complex intrinsic abnormalities in osteoblast lineage cells of X-linked hypophosphatemia: Analysis of human iPS cell models generated by CRISPR/Cas9-mediated gene ablation.

doi: 10.1016/j.bone.2024.117044

Figure Lengend Snippet: Fig. 8. Enhanced phosphorylation of CREB and increased expression of PTHRP in osteoblast lineage cells differentiated from PHEX-KO #1 iPSCs. (A-D) PHEX-KO #1 and isogenic control iPSCs were induced to differentiate into the osteoblast lineage, and cells were harvested at the indicated time points to examine the phosphorylation of FRS2α at Tyr196 (A), ERK1/2 at Tyr180/Tyr182 (B), CREB at Ser133 (C), and Smad1 and Smad5 at Ser463 and Ser465 (D) by Western blotting. The results of densitometry are shown in the bottom graphs. (E) Real-time PCR for the expression of PTHRP in osteoblast lineage cells derived from PHEX-KO #1 and isogenic control iPSCs. (F) The expression of Pthrp in osteoblast (OB)-rich and osteocyte (OCy)-rich cells isolated from WT and Hyp mice. Data in graphs are shown as the mean ± SD (n = 3). #, P < 0.05; ##, P < 0.01 vs Day 14. *, p < 0.05.

Article Snippet: After blocking with Blocking One P reagent (Nacalai Tesque Inc.) or Block Ace reagent (Dainippon Pharmaceuticals, Osaka, Japan), the membranes were incubated at 4 ◦C overnight with the following primary antibodies: antiDMP1 rabbit polyclonal antibody (TaKaRa, Shiga, Japan), anti-OPN rabbit polyclonal antibody (ProteinTech, Rosemont, IL, USA), antiPiT1 (SLC20A1) rabbit polyclonal antibody (H-130; Santa Cruz Biotechnology, Santa Cruz, CA), anti-SLC20A2 (PiT-2) rabbit polyclonal antibody (ProteinTech), anti-GAPDH goat polyclonal antibody (V-18; Santa Cruz Biotechnology), anti-phosphorylated ERK1/2 antibody, antiERK1/2 antibody, anti-phosphorylated CREB (Ser133) antibody, antiCREB antibody, anti-phosphorylated FRS2α antibody (Cell Signaling Technology, Beverly, MA, USA), and anti-FRS2α antibody (Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Expressing, Control, Western Blot, Real-time Polymerase Chain Reaction, Derivative Assay, Isolation

Journal: iScience

Article Title: Cell-type specific circadian transcription factor BMAL1 roles in excitotoxic hippocampal lesions to enhance neurogenesis

doi: 10.1016/j.isci.2024.108829

Figure Lengend Snippet:

Article Snippet: Phospho-CREB (Ser133) (87G3) Rabbit mAb , Cell Signaling , Cat#14001; RRID: AB_2798359.

Techniques: TUNEL Assay, Quantitative RT-PCR, Software

Journal: Cell Communication and Signaling : CCS

Article Title: Local GHR roles in regulation of mitochondrial function through mitochondrial biogenesis during myoblast differentiation

doi: 10.1186/s12964-023-01166-5

Figure Lengend Snippet: Local GHR regulates mitochondrial biogenesis via IGF1-PI3K/AKT/CREB pathway. a The PPI network of human GHR, IGF1, AKT, CREB and PGC1α. b The PPI network of mouse GHR, IGF1, AKT, CREB and PGC1α. c The PPI network of pig GHR, IGF1, AKT, CREB and PGC1α. d The PPI network of chicken GHR, IGF1, AKT, CREB and PGC1α. Protein–protein interaction was performed by the String database and visualized by Cytoscape (version 3.4.0). e and f Western blots with anti-JAK2, anti-p-JAK2, anti-AKT1, anti-p-AKT1, anti-CREB1, anti-p-CREB1 and anti-β-actin at 48 h after transfection with si- GHR and si-NC. g and h Western blots with anti-AKT1, anti-p-AKT1, anti-CREB1, anti-p-CREB1 and anti-β-actin at 48 h after transfection with si- IGF1 and si-NC. i and j Western blots with anti-AKT1, anti-p-AKT1, anti-CREB1, anti-p-CREB1 and anti-β-actin at 48 h after co-transfection with si- GHR + pcDNA3.1- IGF1 , si- GHR + pcDNA3.1 and si-NC + pcDNA3.1. k LY294002 (PI3K inhibitor) was added at 24 h before measuring the expression of PGC1α by RT-qPCR at 48 h after transfection of pcDNA3.1- IGF1 and pcDNA3.1. l GSK690693 (AKT inhibitor) was added at 24 h before measuring the expression of PGC1α by RT-qPCR at 48 h after transfection of pcDNA3.1- IGF1 and pcDNA3.1. m and n LY294002 or GSK690693 was added at 24 h before measuring the protein levels with anti-PGC1α, anti-CREB1, anti-p-CREB1 and anti-β-actin at 48 h after transfection of pcDNA3.1- IGF1 and pcDNA3.1. o The expression of CREB and PGC1α was measured by RT-qPCR at 48 h after transfection with pcDNA3.1- CREB and pcDNA3.1. p and q Western blots with anti-PGC1α, anti-CREB1, anti-p-CREB1 and anti-β-actin at 48 h after transfection with pcDNA3.1- CREB and pcDNA3.1. r Dual-Luciferase report assays transfected with reporter vectors containing different length of 5′ upstream region of PGC1α . s Dual-Luciferase report assays of CREB overexpression co-transfected with reporter vectors containing different length of 5′ upstream region of PGC1α. Data are shown as mean ± SEM, * p < 0.05, ** p < 0.01

Article Snippet: The antibodies and their dilutions utilized for western blots were as follow: anti-GHR (bs-0654R; Bioss, China; 1:500), anti-PGC1α (bs-1832R; Bioss, China; 1:500), anti-NRF1 (12,482–1-AP; Proteintech, USA; 1:500), anti-TOMM20 (AF1717; Beyotime, China; 1:500), anti-JAK2 (bs-0908R; Bioss, China; 1:1000), anti-p-JAK2 (bsm-52171R; Bioss, China; 1:1000), anti-AKT1 (bs-0115 M; Bioss, China; 1:500), anti-p-AKT1 (66,444–1-Ig; Proteintech, China; 1:500), anti-CREB1 (bs-0035R; Bioss, China; 1:500), anti-p-CREB1 (bs-0036R; Bioss, China; 1:500), anti-β-actin (bs-0061R; Bioss, China; 1:1000), goat anti-rabbit IgG-HRP (bs-0295G; Bioss, China; 1:5000), goat anti-mouse IgG-HRP (bs-0296G; Bioss, China; 1:5000).

Techniques: Western Blot, Transfection, Cotransfection, Expressing, Quantitative RT-PCR, Luciferase, Over Expression

Journal: Cell Communication and Signaling : CCS

Article Title: Local GHR roles in regulation of mitochondrial function through mitochondrial biogenesis during myoblast differentiation

doi: 10.1186/s12964-023-01166-5

Figure Lengend Snippet: Schematic diagram for the mechanistic model of the GHR roles in regulation of mitochondrial function during myoblast differentiation. Local GHR enhances mitochondrial function by promoting mitochondrial biogenesis via IGF1 -PI3K/AKT/CREB pathway during myoblast differentiation. This graphical abstract was created with Biorender.com

Article Snippet: The antibodies and their dilutions utilized for western blots were as follow: anti-GHR (bs-0654R; Bioss, China; 1:500), anti-PGC1α (bs-1832R; Bioss, China; 1:500), anti-NRF1 (12,482–1-AP; Proteintech, USA; 1:500), anti-TOMM20 (AF1717; Beyotime, China; 1:500), anti-JAK2 (bs-0908R; Bioss, China; 1:1000), anti-p-JAK2 (bsm-52171R; Bioss, China; 1:1000), anti-AKT1 (bs-0115 M; Bioss, China; 1:500), anti-p-AKT1 (66,444–1-Ig; Proteintech, China; 1:500), anti-CREB1 (bs-0035R; Bioss, China; 1:500), anti-p-CREB1 (bs-0036R; Bioss, China; 1:500), anti-β-actin (bs-0061R; Bioss, China; 1:1000), goat anti-rabbit IgG-HRP (bs-0295G; Bioss, China; 1:5000), goat anti-mouse IgG-HRP (bs-0296G; Bioss, China; 1:5000).

Techniques: